Klebsiella pneumoniae sequence type 147: a high-risk clone increasingly associated with plasmids carrying both resistance and virulence elements

Abstract Introduction. The first hybrid resistance/virulence plasmid, combining elements from virulence plasmids described in hypervirulent types of Klebsiella pneumoniae with those from conjugative resistance plasmids, was described in an isolate of sequence type (ST) 147 from 2016. Subsequently, this type has been increasingly associated with these plasmids. Hypothesis or gap statement. The extent of carriage of hybrid virulence/resistance plasmids in nosocomial isolates of K. pneumoniae requires further investigation. Aim. To describe the occurrence of virulence/resistance plasmids among isolates of K. pneumoniae received by the UK reference laboratory, particularly among representatives of ST147, and to compare their sequences. Methodology. Isolates received by the laboratory during 2022 and the first half of 2023 (n=1278) were screened for virulence plasmids by PCR detection of rmpA/rmpA2 and typed by variable-number tandem repeat analysis. Twenty-nine representatives of ST147 (including a single-locus variant) from seven hospital laboratories were subjected to long-read nanopore sequencing using high-accuracy q20 chemistry to provide complete assemblies. Results.rmpA/rmpA2 were detected in 110 isolates, of which 59 belonged to hypervirulent K1-ST23, K2-ST86 and K2-ST65/375. Of the remainder, representatives of ST147 formed the largest group, with 22 rmpA/rmpA2-positive representatives (out of 47 isolates). Representatives were from 19 hospital laboratories, with rmpA/rmpA2-positive isolates from 10. Nanopore sequencing of 29 representatives of ST147 divided them into those with no virulence plasmid (n=12), those with non-New Delhi metallo-β-lactamase (NDM) virulence plasmids (n=6) and those carrying blaNDM-5 (n=9) or blaNDM-1 (n=2) virulence plasmids. These plasmids were of IncFIB(pNDM-Mar)/IncHI1B(pNDM-MAR) replicon types. Most of the non-NDM virulence plasmids were highly similar to the originally described KpvST147L_NDM plasmid. Those carrying blaNDM-5 were highly similar to one another and to previously described plasmids in ST383 and carried an extensive array of resistance genes. Comparison of the fully assembled chromosomes indicated multiple introductions of ST147 in UK hospitals. Conclusion. This study highlights the high proportion of representatives of ST147 that carry IncFIB(pNDM-Mar)/IncHI1B(pNDM-MAR) hybrid resistance virulence plasmids. It is important to be aware of the high probability that representatives of this type carry these plasmids combining resistance and virulence determinants and of the consequent increased risk to patients.


INTRODUCTION
Klebsiella pneumoniae is an important nosocomial pathogen increasingly associated with resistance and, at least among some community-acquired types, with hypervirulence [1][2][3].The latter, predominantly belonging to sequence types (STs) 23, 86 and 65 of capsular types K1 and K2, carry virulence plasmids containing siderophores, heavy metal resistance genes and regulators of mucoviscosity genes rmpA/rmpA2 [4,5].These plasmids are generally non-conjugative, but in some cases have combined with conjugative resistance plasmids to become conjugative hybrid resistance/virulence plasmids [6,7].As a consequence, virulence plasmids are no longer confined to 'hypervirulent' types and appear in other types, including 'highrisk' clones such as STs 11,14,15,383 and 147, which often carry resistance genes, particularly carbapenemase genes [8][9][10].The first hybrid virulence/resistance plasmid was described in an isolate of K. pneumoniae ST147 collected in 2016, which also carried bla NDM-1 in a separate plasmid [11].Subsequently, isolates carrying both bla NDM-5 and virulence elements in the same plasmid were described [12,13].At that time, hybrid resistance/virulence plasmids were remarkable, but they are now becoming common, especially, in our experience, among representatives of ST147.This is in a type already regularly found with at least one carbapenemase, typically NDM and/or OXA-48-like [14,15]; it has even been described with three (KPC-2, NDM-1 and OXA-48) [16].
Other countries have also reported ST147 carrying hybrid virulence/resistance plasmids; for example, there has been a very large outbreak of NDM-1-producing ST147 in Tuscany, Italy with the outbreak strain carrying a large hybrid virulence/resistance plasmid [17][18][19].In that case the bla NDM-1 was in a separate plasmid.Examples have also been reported in Switzerland, Russia and Egypt [20][21][22][23].Here we describe the occurrence of hybrid virulence/resistance plasmids among isolates received by the UK Health Security Agency national reference laboratory for typing and highlight the increasing proportion of representatives of ST147 that carry them.This work was done using long-read nanopore sequencing with q20 chemistry, which provides single-molecule accuracy of at least 99 % [24], allowing complete assembly of isolates and providing a powerful tool for describing these plasmids.
Isolates subjected to nanopore sequencing were labelled by a unique number (prefixed by KP) followed by a hospital code, the week and year of receipt by the laboratory, their sequence type and the carbapenemase genes that they carried (e.g.KP2_L3_37.21_ST147_NDM5).Hospitals were labelled according to the region in England from which they were received and the number within that region (L, London; SE, South East England; WM, West Midlands; YH, Yorkshire and Humber).
This study was part of a larger one that involved sequencing a further 98 isolates of K. pneumoniae.Where relevant, isolates belonging to other sequence types (ST395, ST2096 and ST1558) that carried virulence plasmids are mentioned; these were sequenced in exactly the same way as those belonging to ST147.These sequences are also available under project PRJNA1010831.

Chromosome comparisons
Fully assembled chromosomes were compared by split kmer analysis (SKA) with the tool 'SKA' (https://github.com/bacpop/ska.rust, version 2 release v0.3.2) developed by Harris [31] with a kmer size of 31.Alignment was created against the chromosome of KP96_L17_12.23_ST6796_NDM5(identified as having the lowest median mash distance from the others) using SKA map with 'ambig mask' option.This alignment was used to create a tree with Gubbins [32] v3.3.1 (which corrects for recombination) using default settings and 1000 bootstraps.

Isolates carrying virulence plasmids
During 2022 and the first half of 2023, 1278 non-duplicate patient isolates of K. pneumoniae were referred to the laboratory for typing from 97 hospital laboratories to inform cross-infection investigations and/or to detect 'hypervirulence' elements.The majority (49 %) were from screening (from rectal swabs, faeces, nasal and pooled swabs), but they also included those from blood (

Chromosome comparison
Since the sequences for most of these isolates (28/29) were fully assembled into closed chromosome and plasmid contigs, this afforded the possibility of comparing the complete chromosome sequences in the absence of the plasmid sequences (Fig. 3).The comparison included chromosome assemblies from two different runs carried out on the same extract for one of the isolates (KP124_L5_21.23_ST147_NDM5)and from runs on two different extracts (from different picks) for another of the isolates (KP51_L9_46.22_ST147_NDM1_OXA48)to assess the potential variation expected for an isolate.These different runs clustered most closely with the other run of the pair.The comparison revealed that the chromosomes of the two isolates from neonates on the same ward (KP10_L3_11.22_ST147and KP11_L3_11.22_ST147)clustered very closely together, as did the chromosomes of those that also carried bla NDM-14 plasmids (KP86_L3_06.23_ST147_NDM14,KP87_L3_06.23_ST147_NDM14 and KP88_L3_06.23_ST147_NDM14), also from that hospital group but clearly distinct from the neonatal isolates.The chromosomes of all of the isolates carrying bla NDM-5 virulence plasmids were all in the same broad clade.These isolates did not cluster according to hospital, showing that isolates of this widely found high-risk clone from the same hospital are not necessarily closely linked.However, the chromosomes of KP13_L3_16.22_ST147_NDM1 and KP15_L3_19.22_ST147_NDM1 from the same hospital (L3) did cluster with one another.The chromosomes of the isolates that did not carry virulence plasmids clustered apart from those that did, but mostly were not closely related.However, in addition to the aforementioned neonatal isolate cluster, two isolates from hospital WM3 clustered together (KP63_WM3_49.22_ST147_OXA181and KP79_WM3_03.23_ST147_OXA181).Interestingly, the chromosome of KP29_L6_38.22_ST147_NDM5 was an outlier from the rest; this isolate differed from the others in that the assembly put the bla NDM gene (bla NDM-5 in this case) in the chromosome rather than in a plasmid; this was the case in two separate runs done on this isolate.
While there was clear evidence for limited transmission between patients among the set, fortunately, we did not observe the rapid and extensive spread observed by Martin et al. and others in the outbreaks of K. pneumoniae ST147 in Italy [18,35].Nevertheless, that these isolates are widely found among UK hospitals and that they often carry conjugative hybrid resistance/virulence plasmids is extremely worrying.Moreover, we have observed a clear link between the bla NDM-5 hybrid plasmids found in isolates of ST147 and those in ST383, both from the UK and from Spain, highlighting their capacity to move to other high-risk clones.
While it is unlikely that these isolates that carried hybrid virulence/resistance plasmids display the full virulence characteristics associated with the typical hypervirulent types such as K1-ST23 and K2-ST86, 5 of the 29 isolates sequenced were from blood, indicating invasive disease.One of these (KP39_L11_43.22_ST147_NDM5)was received as a query hypervirulent strain, suggesting a virulent phenotype.All but one (KP119_WM3_21.23_ST147_NDM1)also carried genes encoding yersiniabactin in the chromosome, also regarded as a virulence factor, but that is not unusual among currently circulating clinical isolates of K. pneumoniae.Studies on isolates from the outbreak in Tuscany showed that they did not exhibit the full hypervirulent phenotype when tested in the Galleria mellonella infection model and in a subcutaneous model of infection in immunocompetent CD1 mice.They showed variably enhanced serum resistance in serum bactericidal assays [18,19], depending on pal, csrD and ramR genes encoding surface components in the chromosome, with inactivation of CsrD associated with increased capsule production and thickness and a consequent increased serum fitness.
In common with other studies, our isolates were divided into distinct K/KL types.Six of the 29 isolates sequenced, all lacking virulence plasmids, belonged to the KL10 type, described as being predominantly found in Asian countries and associated with NDM and OXA-48-like carbapenemases [15].However, most of the isolates (22/29), including all those carrying bla NDM-5 virulence plasmids belonged to the KL64 type, associated with Europe [10].One isolate belonged to the K20 capsular type; this seems to be more rarely found but has been described among isolates from Switzerland [20] and Russia [21].
Fig. 3. Comparison of the fully assembled chromosomes from isolates in this study.Comparison was by split kmer analysis (SKA) [31].Chromosomes were aligned against the chromosome of KP96_L17_12.23_ST6796_NDM5and a tree created with Gubbins [32].The tree was rooted on the KP29_ L6_38.22_ST147_NDM5outlier.Isolates are labelled with a unique KP number followed by a hospital code, receipt date (week.year),sequence type (ST) and any carbapenemase genes carried (in any contig).Isolates are coloured according to whether they had no hybrid virulence plasmid (in blue), a non-NDM hybrid virulence plasmid (in grey and in green), those with a hybrid virulence resistance plasmid carrying bla NDM-1 (highlighted in gold) and those with a hybrid virulence resistance plasmid carrying bla NDM-5 (highlighted in crimson), as in Table S1.The comparison is of the closed chromosome contigs alone.Bootstrap values shown are from 1000 replicates.Results from two separate runs are included on two isolates (KP51_L9_46.22_ST147_NDM1_OXA48 and KP124_L5_21.23_ST147_NDM5).
The two isolates neonates described here were unusual in that they carried no carbapenemase genes, unlike almost every other isolate of this type received by the UK reference laboratory.All the isolates, even these relatively susceptible ones, carried an extensive array of resistance genes.For example KP99_L14_14.23_ST147_NDM5_OXA48carried aph(3')-Ia, aadA1, aph(3')-VI, aac(6')-Ib, bla CTX-M-15 , bla TEM-1B/C , bla OXA-9 , mph(A), truncated catA1, qnrS1, sul1 and dfrA5, in addition to bla NDM-5 in the NDM-5 hybrid virulence/resistance plasmid, aph(6)-Id, aph(3'')-Ib, aph(3')-VIb and bla CTX-M-14b , in addition to bla OXA-48 in the OXA-48 plasmid, and rmtF, aac(6')-Ib-Hangzhou, ARR-2, fosA, oqxAB and bla SHV-11/67 in other plasmids and the chromosome (nearest alleles given where there is not an exact match).We are not aware of colistin resistance among the representatives described here but note the experience in Tuscany, where a colistin and tigecycline-resistant variant evolved during the outbreak as a result of mutations in RamR and MgrB proteins, resulting in premature stop codons and associated with tigecycline and colistin resistance, respectively [36].We have previously reported colistin resistance in representatives of ST147 that was not associated with mcr genes (MIC 8 mg l −1 ) [11], as have others [16,18], with Di Pilato et al. also noting mutations in chromosomal genes associated with colistin resistance (pmrA, crrB, mgrB) among representatives of ST147.Phenotypic susceptibility testing on isolates of ST147 has been reported by many authors and reflects the substantial complement of resistance genes that they carry [11,16,18,19,23].
Isolates received by the national reference laboratory will not have been without bias; the mere fact that they were received for typing means that they will have been biased towards incidents of suspected or actual cross-infection.Some hospitals choose to send isolates while others do not.Nevertheless, the results can identify trends, and the finding of virulence plasmids in representatives of ST147 from multiple hospital laboratories (19) and regions (4) implies a widespread issue.
In conclusion, this study has shown that hybrid virulence resistance plasmids in K. pneumoniae were strongly associated with ST147 among referrals to the UK reference laboratory during 2022/3.In terms of numbers, ST147 was second only to K1-ST23 (the archetypal hypervirulent type) in being the type most associated with virulence plasmids among isolates referred to us (there were 49 isolates of K1-ST23 and 22 of ST147 carrying virulence plasmids on the basis of rmpA/rmpA2 screening).In ST147, these are conjugative plasmids combining virulence and resistance elements.Almost half of representatives of this type carried these plasmids.Most worryingly, a hybrid plasmid carrying bla NDM-5 was widely found among representatives and has also been described in representatives of ST383 and of ST1558, highlighting a wide distribution and the potential for spread between types.These observations show that ST147 should be considered a high-risk clone for carriage of these plasmids.Description of these plasmids was greatly facilitated by nanopore sequencing, which enabled complete assemblies of both the chromosomes and plasmids of these isolates.

Table 1 .
Details of representatives of ST147 and ST6796 sequenced, arranged by hospital group and region and summary of virulence and carbapenemase plasmids found.Details of resistance and virulence genes and other plasmids found are provided in TableS1, available in the online version of this article.Hospitals are labelled according to region in England and number within that region
[33]he remainder, it was striking that representatives of ST147 formed the biggest group, with 22 rmpA/rmpA2-positive representatives (out of 47 isolates) (47 %), followed by ST14/2096 (5 out of 48 representatives).Other high-risk clones were not well represented among rmpA/rmpA2 positive isolates -there were just two isolates of ST101 and one of ST383.Four (of 17) representatives of ST395 carried rmpA/rmpA2.Others have also recently described representatives of ST395 carrying hypervirulence plasmids[33].Representatives of ST147 were from 19 hospital laboratories, with rmpA/rmpA2-positive isolates received from 10 of them.